title: Characterization of monomeric substates of ascorbate oxidase.
abstract: Ascorbate oxidase (AAO) is a large, multidomain, dimeric protein whose folding/unfolding pathway is characterized by a complex, multistep process. Here we used fluorescence correlation spectroscopy to demonstrate the formation of partially folded monomers by pH-induced full dissociation into subunits. Hence, the structural features of monomeric AAO could be studied by fluorescence and CD spectroscopy. We found that the monomers keep their secondary structure, whereas subtle conformational changes in the tertiary structure become apparent. AAO dissociation has also been studied when unfolding the protein by high hydrostatic pressure at different pH values. A strong protein concentration dependence was observed at pH 8, whereas the enzyme was either monomeric or dimeric at pH 10 and 6, respectively. The calculated volume change associated with the unfolding of monomeric AAO, ΔV ∼ -55 mL·mol(-1), is in the range observed for most proteins of the same size. These findings demonstrate that partially folded monomeric species might populate the energy landscape of AAO and that the overall AAO stability is crucially controlled by a few quaternary interactions at the subunits' interface.
authors.affiliation: NAST Centre, University of Rome, Tor Vergata, Rome, Italy.
authors.forename: Almerinda
authors.initials: A
authors.lastname: Di Venere
authors.forename: Eleonora
authors.initials: E
authors.lastname: Nicolai
authors.forename: Nicola
authors.initials: N
authors.lastname: Rosato
authors.forename: Antonello
authors.initials: A
authors.lastname: Rossi
authors.forename: Alessandro
authors.initials: A
authors.lastname: Finazzi Agrò
authors.forename: Giampiero
authors.initials: G
authors.lastname: Mei
mesh_terms: D001204:Ascorbate Oxidase / Q000737:chemistry / Q000378:metabolism*; D002942:Circular Dichroism; D006863:Hydrogen-Ion Concentration; D017433:Protein Structure, Secondary; D013050:Spectrometry, Fluorescence
chemical_list: D001204:Ascorbate Oxidase